Transformation is a commonly-employed genetic engineering procedure in which new genetic material is acquired by eukaryotic or procaryotic cells by the incorporation of exogenous DNA sequences coding for a desired protein or polypeptide. Ordinarily, the number of cells in a population undergoing transformation which actually incorporate the exogenous DNA is quite low. The problem of low transformation of the exogenous DNA can be obviated by transforming the host cell with a selection marker, (i.e. DNA encoding an easily-identifiable marker, such as resistance to an antibiotic) in addition to the exogenous DNA sequence. Upon transformation, the cell population is examined for the presence of the marker. Depending upon whether and how closely the selection marker is linked to the exogenous protein-encoding DNA, cells carrying the selection marker will also contain the exogenous DNA. Those cells which have successfully incorporated the marker DNA will exhibit the marker identity (e.g. survival in media containing the antibiotic) and those cells which have failed to incorporate the marker will not exhibit the marker feature (e.g. they will die upon exposure to the antibiotic).
The level of exogenous protein expressed by the transformed cells can also be substantially increased where a DNA encoding an amplifiable gene as well as a selectable marker is included in the transformation process. Amplification of a gene involves exposing the transformed cell to environmental pressure sufficient to require the cell to produce more copies of the amplifiable gene for survival.
The marker/amplification system most extensively used employs the gene for dihydrofolate reductase (DHFR), a fairly ubiquitous gene found in many cell lines. Exposing a cell transformed with DHFR-encoding DNA to cytotoxic concentrations of methotrexate (MTX) encourages the cell to amplify DHFR to survive. Cells which survive the MTX selection procedure have many copies of the DNA encoding DHFR. When the DHFR gene is on a plasmid containing a DNA sequence for another gene, that gene generally becomes amplified as well. Thus when transforming a cell with a vector containing a DHFR gene and an exogenous gene, the DHFR behaves as a selectable marker to enable the identification of those cells which have incorporated the vector from those cells which have not. The DHFR itself is also capable of being amplified and consequently amplifies the exogenous DNA. In practice, the DHFR system has demonstrated general utility only with one cell line, a Chinese hamster ovary line which is deficient in DHFR (CHO DHFR.sup.-). [Urlaub et al., Proc. Natl. Acad. Sci. U.S.A., 77:4216-4220 (1982)]. Some efforts have been reported to overcome this limitation. [See e.g., Simonson, C. C. et al., Proc. Natl. Acad. Sci, U.S.A., 80:2495-99, (1983), Murray, M. J. et al., Mol. Cell, Biol. 3:32-43 (1983 )]. However, obtaining the optimal conditions necessary for expression of exogenous proteins has yet proven difficult.
Other selectable, amplifiable markers include adenosine deaminase ("ADA") [See, e.g., R. J. Kaufman et al., Proc. Nat'l. Acad. Sci. USA, 83:3136-3140 (1986) and those listed in G. R. Stark et al., "Gene Amplification" in Ann. Rev. Biochem, 53: 447-491 (1984). Other systems for amplifying and expressing heterologous DNA in a variety of different cell lines remain an unfulfilled need in the art.